Use of an enzyme inhibitor to retard the degradation of gelatin



United States Patent O 3 503,746 USE OF AN ENZYME INHIBITOR T RETARD THE DEGRADATION 0F GELATIN James A. McClintock, Rochester, N.Y., assignor to Eastman Kodak Company, Rochester, N.Y., a corporation of New Jersey No Drawing. Filed May 3, 1967, Ser. No. 635,646

Int. Cl. G03c 1/06 US. Cl. 96-94 14 Claims ABSTRACT OF THE DISCLOSURE Aqeous solutions of gelatin and photographic gelatinosilver halide emulsions containing L-l-tosylamido-Z-phenethyl-chloromethyl-ketone to control enzyme activity and decelerate bacterial growth and reproduction. A phenolic biostat may optionally be added for improved results.

BACKGROUND OF THE INVENTION Field of the invention This invention relates to controlling enzyme activity and thereby decelerating bacterial growth and reproduction in gelatin and the compositions obtained thereby. Another aspect of the invention relates to silver halide emulsions and photographic elements in which enzyme activity and continued bacterial growth and reproduction is controlled.

Description of the prior art It is well known that photographic gelatin is a chemically stable substance when stored in a warm aqueous emulsion under aseptic conditions. However, an aqueous gelatin emulsion which is contaminated with pathogenic bacteria is rapidly degraded by what is known as bacterial action. In such bacterial action gelatin is not directly consumed by the bacteria because of the large size of gelatins polypeptide molecules. Instead, the bacteria generate and inoculate the gelatin sol with a proteolytic enzyme which catalyzes the hydrolysis of gelatin into smaller polypeptides, peptides, and amino acids which are capable of passing through the bacterial cell walls, thus suporting the intracellular metabolism necessary for the organism growth and reproduction. Bacterial action causes serious problems in the photographic industry. Bacterial action causes severe defects in the developed image as it results in sensitized spots which becomes spontaneously developable without image exposure. In addition, bacterial action causes severe fog brought on by degradation of the gelatin. Degradation of gelatin also changes its viscosity and seriously interferes with the anchoring characteristics of thin subbing layers.

It has been generally accepted that gelatin sols and photographic emulsions held for extended periods in cold storage before coating require not only a depressant for slowing down the living processes of organisms, but also a mechanism for destroying or inactivating the proteolytic enzymes already present or being produced by the still viable organisms. If the enzymes are not destroyed or inactivated, they will hydrolyze the gelatin sol.

Unfortunately, most of the more frequently used emulsion biostats at practical concentrations are apt to act more as an enzyme preservative than a depressant. Thus, although bacterial activity is slowed, a minute quantity of enzyme already formed remains active. Pasteurization at 170-180 F. destroys much of the enzyme potential but recontamination does occur and there are systems where pasteurization is inconvenient or impractical.

Some of the compounds useful as gelatin preservatives in the past have been 1,3-diketones including highly enolized 1,3-diketones such as acetyl acetone; formaldehyde, phe- I101, thymol, esters of p-hydroxybenzoic acid, alcohols, and others as described in US. Patent 2,226,183 of Stand et al. issued Dec. 24, 1940; British Patent 987,010 of Du Pont issued Mar. 24, 1965; and British Patent 968,883 of Gevaert issued Sept. 2, 1964. These compounds, however, are directed towards slowing down the living processes of organisms and do very little in inactivating the proteolytic enzymes already present.

There are compounds known to be enzyme inhibitors but they do not all function in an equivalent manner when incorporated into a gelatin composition. For example, I have investigated iodoacetic acid and various fluorides such as sodium fluoride, potassium fluoride and potassium fluosilicate, but in permissible concentrations found their activity to be low in certain gel sols and photographic emulsions and under some conditions they fail entirely.

SUMMARY OF THE INVENTION I have found that L-1-tosylamido-2-phenethyl-chloromethyl-ketone (TPCK) will inhibit the action of enzymes in aqueous gelatin solutions. TPCK can also be incorporated into a gelatino-silver halide emulsion and will control enzyme activity, which degrades the gelatin and is necessary for continued bacteria growth, without adverse sensitomeric effects. In addition, the combination of this enzyme inhibitor with a phenolic biostat further preserves gelatin from biological degradation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS TPCK, which has the following formula 0 If Q-om-pn-o-omor can be used in any concentration effective for the intended purpose. Generally, good results are obtained when the TPCK is employed in a concentration of from about 0.015 to about 0.033 gram per liter of gelatin solution or gelatino-silver halide emulsion.

TPCK is a nown compound as shown, for example, by Zajac and Crowell, Effect of Enzymes on the Interaction of Enteroviruses with Living HeLa Cells, Journal of Bacteriology, March 1965, vol. 89, No. 3, page 575. Its use in gelatin and gelatino-silver halide emulsions has not been described, however.

Phenolic biostats may also be employed in combination With TPCK to give the best results in preserving gelatin. The biostat thus controls the bacteria which produces enzymes and the TPCK inhibits the enzymes from attacking the gelatin. Any of the well-known phenolic biostats can be employed for this purpose such as phenol, thymol, p-chloro-meta-xylenol, etc.

The following example will illustrate the invention but is not to be construed to limit it in any way.

EXAMPLE I To portions of a coarse-grain gelatino-silver bromoodide emulsion containing approximately 9 mole percent iodide and 210 grams of gelatin per silver mole are added TPCK at 0.079 gram per mole of silver 0.026 gram per liter of solution, p-chloro-meta-xylenol (a known phenolic biostat) at 0.088 gram per mole of silver and a mixture of TPCK (0.088 gram per silver, i.e., 0.029 gram per liter of solution mole) and p-chloro-rneta-xylenol (0.088 gram per silver mole). The samples are kept at 47 F. for 7 and 14 Weeks. Since the action of enzymes results in a loss of viscosity, degradation of gelatin can be measured by viscosity changes. The best preservative will have the least viscosity change. After storage, viscosity readings are taken with the following results:

1 Reading taken after holding sample for 24 hours at 98 F. 2 Reading taken after holding sample for 1 hour at 98 F. 3 Sample almost completely hydrolyzed and worthless.

The silver halide emulsion of a photographic element containing the enzyme inhibitor of this invention can contain conventional addenda such as gelatin plasticizers, coating aids, and hardeners such as aldehyde hardeners, e.g., formaldehyde, mucochloric acid, glutaraldehyde bis- (sodium bisulfite), maleic dialdehyde, aziridines, dioxane derivatives and oxypolysaccharides. Spectral sensitize'rs which can be used are the cyanines, merocyanines, complex (trinuclear) cyanines, complex (trinuclear) merocyanines, styryls, and hemicyanines. Sensitizing dyes useful in sensitizing such emulsions are described, for example in US. Patents 2,526,632 of Brooker and White issued Oct. 24, 1950, and 2,503,776 of Sprague issued Apr. 11, 1950. Developing agents can also be incorporated into the silver halide emulsion if desired or can be contained in a contiguous layer. Various silver salts can be used as the sensitive salt such as silver bromide, silver iodide, silver chloride, or mixed silver halides such as silver chlorobromide or silver bromoiodide. The sliver halides used can be those which form latent images predominantly on the surface of the silver halide grains or those which form latent images inside the silver halide crystals such as described in US. Patent 2,592,250 of Davey and Knott issued Apr. 8, 1952.

The silver halide emulsion layer of a photographic element containing the enzyme inhibitor of the invention can contain in combination with gelatin any of the hydrophilic, water-permeable binding materials suitable for this purpose. Suitable materials include colloidal albumin, polyvinyl compounds, cellulose derivatives, acrylamide polymers, etc. The binding agents for the emulsion layer of the photographic element can also contain dispersed polymerized vinyl compounds. Such compounds are disclosed, for example, in US. Patents 3,142,568 of Nottorf issued July 28, 1964; 3,193,386 of White issued July 6, 1965; 3,062,674 of Houck, Smith and Yudelson issued Nov. 6, 1962; and 3,220,844 of Houck, Smith and Yudelson issued Nov. 30, 1965; and include the water-insoluble polymers of alkyl acrylates and methacrylates, acrylic acid, sulfoalkyl acrylates or methacrylates and the like.

The silver halide emulsion of a photographic element containing the enzyme inhibitor of the invention can be coated on a wide variety of supports. Typical supports are cellulose nitrate film, cellulose ester film, polyvinyl acetal film, polystyrene film, poly (ethylene terephthalate) film and related films or resinous materials as well as glass, paper, metal and the like. Supports such as paper which are coated with alpha-olefin polymers, particularly polymers of alpha-olefins containing two or more carbon atoms, as exemplified by polyethylene, polypropylene, ethylene-butene copolymers and the like can also be employed.

The speed of the photographic emulsions containing the enzyme inhibitor of the invention can be further enhanced by including in the emulsions a variety of hydrophilic colloids such as carboxymethyl protein of the type described in U.S. Patent 3,011,890 of Gates, Jr., Miller and of the instant invention can be used in various kinds of photographic systems. In addition to being useful in X-ray and other nonoptically sensitized systems, they can also be used in orthochromatic, panchromatic, and infrared sensitive systems. The sensitizing addenda can be added to photographic systems before or after any sensitizing dyes which are used.

Silver halide emulsions containing the enzyme inhibitor of the invention can be used in color photography, for example, emulsions containing color-forming couplers or emulsions to be developed by solutions containing couplers or other color-generating materials, emulsions of the mixed-packet type such as described in US. Patent 2,698,794 of Godowsky issued Jan. 4, 1955; in silver dyebleach systems; and emulsions of the mixed-grain type such as described in US. Patent 2,592,243 of Carroll and Hanson issued Apr. 8, 1952.

Silver halide emulsions containing the enzyme inhibitor of the invention can be sensitized using any of the wellknown techniques in emulsion making, for example, by digesting with naturally active gelatin or various sulfur, selenium, tellurium compounds and/ or gold compounds. The emulsions can also be sensitized with salts of noble metals of Group VIII of the Periodic Table which have an atomic weight greater than 100.

Silver halide emulsions containing the enzyme inhibitor of the invention can be used in diffusion transfer processes which utilize the undeveloped silver halide in non-image areas of the negative to form a positive by dissolving the undeveloped silver halide and precipitating it on a silver layer in close proximity to the original silver halide emulsion layer. Such processes are described in US. Patents 2,352,014 of Rott, issued June 20, 1944; 2,543,181 of Land, issued Feb. 27, 1951; and 3,020,155 of Yackel, Yutzy, Foster and Rasch, issued Feb. 6, 1962. The emulsions can also be used in diffusion transfer color processes which utilize a diffusion transfer of an image-wise distribution of developer, coupler or dye, from a light-sensitive layer to a second layer, while the two layers are in close proximity to one another. Silver halide emulsions containing the enzyme inhibitor of the invention can be processed in stabilization processes such as the ones described in US. Patent 2,614,927 of Broughton and Woodward, issued Oct. 21, 1952, and as described in the article Stabilization Processing of Films and Papers by H. D. Russell, E. C. Yackel and J. S. Bruce in PSA Journal, Photographic Science and Technique, vol. 16B, October 1950.

Combinations of all the above-mentioned addenda can be used if desired.

Although the invention has been described in considerable detail with particular reference to certain preferred embodiments thereof, variations and modifications can be effected within the spirit and scope of the invention as described hereinbefore and as defined in the appended claims.

I claim:

1. A composition comprising an aqueous solution of gelatin and L-l-tosylamido-2-phenethyl-chloromethylketone.

2. The composition of claim 1 wherein said L-ltosylamido-Z-phenethyl-chloromethyl-ketone is present in a concentration of from about 0.015 to about 0.033 gram per liter of solution,

3. The composition of claim 1 wherein said aqueous solution of gelatin is a photographic gelatino-silver halide emulsion.

4. The photographic emulsion of claim 3 wherein said L-1-tosylamido-2-phenethyl-chlorometliyl-ketone' is present in a" concentration of from about 0.015 to about 0.033 gram per liter of emulsion.

5. The composition of claim 1 which also contains a phenolic "biostat. 5

6. The composition of claim 2 which also contains a phenolicbiostat.

7. The photographic emulsion of claim 3 which also containis ia phenolic biostat.

8. The photographic emulsion of claim 4 which also contains a phenolic biostat.

9. Aphotographic element prepared from the emulsion of claimj 4.

10. Aphotographic element prepared from the emulsion of claim 8.

11. A method of controlling bacteria growth in an aqueous gelatin solution which comprises incorporating therein T,-1-tosylamido-2-phenethyl-chloromethyl-ketone.

12. The method of claim 11 wherein said L-l-tosylamido-2 phenethyl-chloromethyl-ketone is present in a concentration of from about 0.015 to about 0.033 gram per liter of solution.

13. The method of claim 12 wherein said aqueous solution of gelatin is a photographic gelatino-silver halide emulsion.

14. The method of claim 12 wherein a phenolic biostat is also incorporated therein.

1 References Cited UNITED STATES PATENTS 7/1969 Ditzer et a1 9694 OTHER REFERENCES NORMAN G. TORCHIN, Primary Examiner ALFONSO T. SURO PICO, Assistant Examiner US. Cl. X.R. 424321 

